richard murray  (Addgene inc)

Journal: ACS infectious diseases

Article Title: Polyamine-Mediated Sensitization of Klebsiella pneumoniae to Macrolides through a Dual Mode of Action.

doi: 10.1021/acsinfecdis.4c00157

Figure Lengend Snippet: Figure 4. Putrescine inhibits protein synthesis. Protein (deGFP) and mRNA levels were simultaneously monitored fluorometrically (433/ 475 and 610/650 nm, respectively) in black 384-well plates using the NEBExpress cell-free protein expression system in 200 mM HEPES buffer with an engineered plasmid encoding deGFP with a malachite green mRNA aptamer. Results shown are normalized by dividing each well’s RFU deGFP value by the malachite green RFU value and converting into a percentage of the maximal signal observed in the untreated condition. Raw RFU data are found in Figure S7A−F. All error bars denote SEM. A one-way ANOVA analysis followed by a Brown−Forsythe and Welch test assessed the significance of the difference, as indicated on the plot.

Article Snippet: Transcription and translation were measured fluorometrically (excitation/emission wavelengths at 610/650 nm MGapt, 485/525 nm deGFP) using an engineered plasmid that encodes a deGFP protein and a malachite green RNA aptamer (PT7-deGFP-MGapt was a gift from Richard Murray, via Addgene plasmid #67741; http:// n2t.net/addgene:67741).62 The cell-free protein synthesis system reaction mixture was prepared following the manufacturer’s protocol and dispensed as 5 μL aliquots into black 384- well plates.

Techniques: Expressing, Plasmid Preparation